1. Field of the Invention
This invention relates to the production of thrombin, thrombin preparations thereby produced and their application in haemostasis.
Thrombin is a proteolytic enzyme, derived from prothrombin, which converts fibrinogen to fibrin. During the normal clotting of blood the insoluble protein fibrin is formed from fibrinogen by the proteolytic action of thrombin. Blood clots are formed to a large extent of fibrin.
2. Description of the Prior Art
Preparations of both bovine and human thrombin have been used in haemostasis. Preparations of bovine thrombin are currently used topically to control haemorrage from puncture sites or from capillary oozing in surgery. Thrombin (USP) is a sterile freeze-dried powder containing the protein obtained from bovine prothrombin and contains calcium. Freeze-dried thrombin preparations are sold under the Trade Mark THROMBOSTAT marketed by Parke Davis. Liquid thrombin preparations are also known and are the subject of U.S. Pat. Nos. 4,696,812 and 4,965,203. Liquid thrombin preparations are generally favoured in that they require no reconstitution prior to use and can be sold in ready-to-use form. Special buffer systems have been used to provide the stable thrombin compositions of U.S. Pat. Nos. 4,696,812 and 4,965,203.
Conventionally thrombin has been prepared from bovine prothrombin derived from bovine plasma. The prothrombin is converted to thrombin using thromboplastin in the presence of calcium chloride. The thrombin thereby obtained is purified by passing filtered thrombin sequentially through an anion-exchange chromatography column and a cation-exchange chromatography column, followed by a one-step elution from the latter column. The process is depicted schematically in FIG. 1. The product thereby obtained has a specific activity of approximately 1,000 U/mg and exhibits a hazy appearance.
Problems exist with the conventional process due to the formation of insoluble salts on the chromatographic media. Removal of these salts requires routine washing with dilute acid. Frequent washing with acid significantly reduces the life of the media.
In the conventional process referred to herein, 10 units of thromboplastin (measured as N.I.H. units of thrombin produced) convert 1 unit of prothrombin (measured as N.I.H. units of thrombin produced) to 1 N.I.H. unit of thrombin, the concentrations of both thromboplastin and prothrombin being measured by an F.D.A.-approved assay for thrombin. This quanity of thromboplastin causes turbidity and increased protein load on the chromatographic media.
It is an object of the present invention to provide an improved and cost effective process for the production of thrombin which results in a thrombin preparation with increased specific activity relative to thrombin preparations obtained in conventional thrombin production.
It is a further object of the invention to provide a clear thrombin preparation relative to the hazy appearance of the conventional thrombin preparations.
It is a still further object of the invention to provide a thrombin preparation with high specific activity.